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D S Millican

Publications and source records attributed to D S Millican.

6 recordsLinked to original sources
PubMed1998

Specific pregnancy-induced angiotensin II type-1 receptor expression in ovine uterine artery does not involve formation of alternate splice variants or alternate promoter usage.

Recently we reported that pregnancy is associated with a dramatic increase in angiotensin II type-1 receptor (AT1-R; both protein and mRNA) in ovine uterine artery endothelial cells (UAEC), which far exceeds that seen in omental (systemic) arteries. Recent reports also suggest that alternate splicing of AT1-R mRNA may play a role in regulation of AT1-R expression in humans. Herein, we have investigated the possibility of alternate transcript splicing/promoter usage in UAEC from pregnant ewes by 5'-RACE (rapid amplification of cDNA 5'-ends). To provide our control "reference" sequences, we first performed 5'-RACE analysis of AT1-R mRNA transcripts in liver, kidney, and adrenal cortex. Analysis of 17 resultant clones showed exceptional homology, indicating that a single identically spliced mRNA product is observed in all three ovine tissues. Homology of the 5'-untranslated region to that of the human was low (34.2%), but four in-context start/stop codons and the beginning of human exons 1 and 5 were highly conserved. Subsequently we isolated 30 individual clones using UAEC RNA from three pregnant ewes and found no evidence of any sequence formed through unique splicing or promoter usage. We conclude that the pregnancy-induced increase in AT1-R expression unique to UAEC during pregnancy is not mediated by splicing of a unique transcript or unique promoter usage.

Angiotensin II↗
PubMedDate not supplied

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor.

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.

Amino Acid Sequence↗
PubMedDate not supplied

Pregnancy induces an increase in the expression of glyceraldehyde-3-phosphate dehydrogenase in uterine artery endothelial cells.

OBJECTIVE: We determined the effects of pregnancy on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in ovine uterine and omental (systemic control) artery endothelial cells (UAEC, OAEC). We also determined in primary cultures of UAEC the effects of exposure to either exogenous nitric oxide (NO) or angiogenic growth factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and epidermal growth factor [EGF]) on UAEC GAPDH expression. METHODS: We isolated UAEC to high purity from both nonpregnant (NP; n = 4) and pregnant ewes (P; 110-120 days' gestation, n = 4) by limited collagenase dispersion and immediately extracted total RNA. In additional experiments performed in vitro, ovine UAEC isolated from NP ewes and maintained in culture (n = 3) were exposed to 1) 100 mumol/L sodium nitro-prusside for 0, 6, 12, or 24 hours or 2) 10 ng/mL bFGF, VEGF, EGF, or vehicle for 24 hours. Total RNA was then immediately extracted. A partial ovine GAPDH (oGAPDH) cDNA was isolated by reverse transcriptase polymerase chain reaction (RT/PCR) and sequenced. A one-tube semiquantitative RT/PCR amplification assay was established, and GAPDH mRNA was subsequently quantified in all samples of total RNA. The PCR products were separated by size, quantified by Southern hybridization analysis, and normalized to 28S rRNA content. Expression of GAPDH protein was also measured by Western analysis of endothelium-derived protein from omental (n = 14) and uterine (n = 16) arteries from NP and P ewes. RESULTS: Pregnancy was associated with a 4.5-fold increase in GAPDH mRNA levels in UAEC, although in vitro exposure of primary cultures of UAEC from NP ewes to NO or angiogenic growth factors did not significantly change GAPDH mRNA expression. A 1.6-fold increase in GAPDH protein was detected in the uterine artery endothelium of P versus NP ewes, but no corresponding increase was found in omental artery endothelium. CONCLUSION: Pregnancy increases the expression of both GAPDH mRNA and protein in UAEC. Furthermore, the pregnancy-induced increase in GAPDH protein in the endothelium of the uterine artery appears specific, as it is not observed in the omental (systemic) artery. This induction is not, however, reproduced in vitro with exogenous NO or angiogenic growth factor treatment (up to 24 hours).

Animals↗